PROJECT SUMMARY Despite advances in clinical detection, breast cancer (BC) is the second leading cause of cancer-related deaths in women. Importantly, recurrent BC is frequently treatment resistant and has poor survival outcomes. The RON receptor tyrosine kinase (RTK) overexpressed in 50-93% of human BCs and high levels of RON occur across all BC subtypes. RON has been shown to be a predictor of BC recurrence, metastasis and poor prognosis in patients with BC. Overexpression of RON in the mammary epithelium of mice is sufficient to induce highly metastatic BC in 100% of female mice. Further, we have recently made the novel discovery that RON expression is sufficient to drive metabolic reprogramming where glycolysis predominates to meet energy demands despite oxygen availability, however, a connection to RON and metabolism has not been examined. Interestingly, published studies show that aerobic glycolysis in tumors correlates with therapeutic resistance and BC recurrence. Moreover, recent studies show that in breast cancer stem-like cells (BCSCs) a shift towards glycolytic processes over that of oxidative phosphorylation is present. Consistent with this data, our recent data show an important role for RON signaling in supporting BCSC phenotypes. Given that BCSCs are advocated to be the cells that evade therapy and lead to recurrence, RON signaling may support recurrence through BCSCs which we speculate may occur through expression of glycolytic genes. Thus, the long-term goal of this proposal is to understand how RON expression supports BC recurrence and to exploit this mechanism for therapeutic intervention, and the overall objective is to elucidate the mechanisms by which RON signaling supports BCSC phenotypes and to target specific vulnerabilities to reduce BC recurrence. To meet this objective, we will test our central hypothesis that inhibiting glycolytic metabolism in RON expressing BC will diminish BC recurrence by targeting BCSCs. We will first determine the mechanism by which RON drives metabolic reprogramming towards aerobic glycolysis in BC cells, and evaluate the functional significance in BCSCs by evaluating the RON-dependent requirement of c-Myc and HIF1 on glycolytic gene expression, metabolite composition, and functional metabolic flux, and examining the consequences of in BCSC phenotypes. Then, we will examine the susceptibility of RON expressing BC/BCSCs to glycolytic inhibition and ensuing effects on BC recurrence. This will be performed by targeting LDHA, which we believe will induce feedback mechanisms that directly affect upstream steps in glycolysis, making it difficult to meet cellular energy needs via aerobic glycolysis. For recurrence, orthotopically implanted tumors will be surgically resected and recurrent tumors evaluated. LDHA will be targeted to prevent recurrence and to treat recurrence, in separate experiments. In vitro, we will perform assays evaluating BCSC-like phenotypes with LDHA targeting that will allow for consideration of therapeutic potential in eradicating tumor-reconstituting cells, a clinically relevant metric.